1. Technical Field of the Invention
The present invention relates to a novel fusion protein with the formula X—Y, or Y—X, wherein X represents a first immunoregulating polypeptide and Y represents a second immunoregulating polypeptide different from X. The present invention also relates to a nucleic acid molecule encoding such a fusion protein and a vector comprising such a nucleic acid molecule. The present invention also provides infectious viral particles and host cells comprising such a nucleic acid molecule or such a vector as well as a process for producing such infectious viral particles. The present invention also relates to a method for recombinantly producing such a fusion protein. Finally, the present invention also provides a pharmaceutical composition comprising such a fusion protein, a nucleic acid molecule, a vector, infectious viral particles and a host cell as well as the therapeutic use thereof.
The present invention is particularly useful in the field of gene therapy and immunotherapy, especially for treating or preventing a variety of diseases, including cancers and infectious diseases (bacteria and virus infections).
2. Description of Background and/or Related and/or Prior Art
Broadly speaking, host's immune responses fall into two categories nonspecific (or innate) and specific (or adaptive or acquired). The differences between these is that an specific immune response is highly specific for a particular antigen whereas nonspecific response does not rely on a repeated exposure to a given pathogen/antigen. The networks controlling the immune system rely on secreted proteins (e.g., cytokines) to turn on and off the functions of immune cells as well as to regulate their proliferation and to control the magnitude of the immune response. Specifically, two types of lymphocytes—B and T cells—are at the core of specific immunity. Upon being triggered by an antigen, B cells divide and the daughter cells synthesize and secrete antibody molecules (humoral immunity). T cell activation entails development of cell-mediated immunity, mediated among others by cytotoxic T lymphocytes (CTL) that specifically eliminates non-self antigen-bearing target cells (e.g., infected or tumoral cells). Activation of a specific (or adaptative) immune response is orchestrated by numerous cytokines. Of particular importance are interleukin (IL)-1, IL-2, IL-6, IL-7, IL-15 and interferon gamma (IFNg). On the other hand, nonspecific (innate) responses involve different types of immune cells, including natural killer (NK) cells, Natural Killer T cells (NKT), dendritic cells (DCs) and macrophages, and are among others mediated by the secretion of cytokines such as IL-2, IL-12, IL-15, IL-18 and IL-21. In reality, however, a strict distinction between specific and nonspecific immune responses is somewhat arbitrary, as the elimination of pathogens and tumors in vivo is likely to involve both types of immune responses acting in concert. Also, through cytokine signalling pathways, specific effectors may play a major role in the induction and activation of nonspecific effectors and vice versa. For example, one striking property of NKT cells is their capacity to rapidly produce large amounts of cytokines in response to T-cell receptor engagement, suggesting that activated NKT cells can also modulate specific immune responses. For a general discussion of immune response, immune effector cells and immune mediators, see for example the most updated editions of “Encyclopedia of Immunology” (Edited by Ivan Roitt and Peter Delves; Academic Press Limited) and “Fundamental Immunology (e.g., 2nd edition, Edited by W. Paul; Raven Press).
It is generally accepted that cancer is a multistep process which results from a loss of the control of cell multiplication. An extensive body of research exists to support the involvement of tumor-associated antigens (TAAs) in the onset of the malignant phenotype. These antigens include oncogene products (e.g., p53, ras, neu, erb), reactivated embryonic gene products (e.g., P91A found in P815 mastocytoma), modified self-antigens (e.g., hyperglycosylated MUC-1), oncogenic viral genes (e.g., early antigens of papillomavirus) and a variety of others. With regard to the mechanism that operates in the recognition and elimination of tumor cells, it has been shown that T lymphocytes play a key role in conferring specificity to tumor rejection. In particular, CD8+ cytotoxic T lymphocytes (CTL) were identified as important effector cells for recognizing specific tumor antigens. CTLs can kill tumors only after they have been presensitized to a tumor antigen and only when it is presented at the cell surface by MHC class I gene products. In many cases, the induction of the anti-tumoral response is also dependent on the presence of CD4+ T cells. In addition to these specific immune effector cells, roles have been identified in tumor rejection for NK cells and other nonspecific effector cells such as NKT and macrophages, which can lyse tumor cells in a manner that is not antigen-dependent and not MHC-restrited.
Despite the fact that the vast majority of tumor-associated antigens is capable of being recognized as foreign by the immune system of the patient and the abundance of tumoricidal immune mechanisms, most cancers do not provoke immunological responses sufficient to control the growth of malignant cells. Tumor cells have developed several mechanisms which enable them to escape host immunity due to a reduction in antigen presentation by the tumor cells or due to a generalized decline in patient's immunity. As the expression of MHC class I determinants on cell surface is essential for the recognition of foreign antigens by CTLs, suppression or failure to express MHC class I antigens is one of the documented mechanisms used by tumor cells to evade the immune system (Tanaka et al., 1988, Ann. Rev. Immunol. 6, 359-380). Another mechanism of immune anergy involves the shedding of tumor antigens, thus preventing the interaction of the immune cells with the tumor target cell itself. Moreover, tumors can activate immunosuppressive molecules to dampen the vigor of immune responses to tumor antigens or to activate apoptosis of immune effector cells. For example, IL-2 may have in some circumstances, a critical role in the maintenance of peripheral tolerance. As a result of its pivotal role in activation-induced cell death (AICD), the T cells generated in response to tumour vaccines containing IL-2 may interpret the tumor cells as self and the tumor-reactive T cells may be killed by AICD-induced apoptosis (Lenardo, 1996, J. Exp. Med. 183, 721-724). Furthermore, IL-2 maintains CD4+ CD25+ negative regulatory T cells and has been reported to terminate CD8+ memory T cell persistence (Shevach, 2000, Ann. Rev. Immunol. 18, 423-449).
A number of studies have documented a critical role for tumor-specific CD4(+) cells in the augmentation of immunotherapeutic effector mechanisms. However, chronic stimulation of such CD4(+) T cells often leads to the up-regulation of both Fas and Fas ligand, and coexpression of these molecules can potentially result in activation-induced cell death (AICD) and the subsequent loss of anti-tumor response. By contrast, resistance to AICD significantly enhances T cell effector activity (Saff et al. 2004, J. Immunol. 172, 6598-6606).
A number of previous approaches have used cytokines to enhance host's immunity, and thus to overcome tumor-induced state of immune anergy. For example, human IL-2 (Proleukin) is an approved therapeutic for advanced-stage metastatic cancer. However, the systemic administration of cytokines is often poorly tolerated by the patients and is frequently associated with a number of side-effects including nausea, bone pain and fever (Mire-Sluis, 1993, TIBTech vol. 11; Moore, 1991, Ann Rev Immunol. 9, 159-191). These problems are exacerbated by the dose levels that are required to maintain effective plasma concentrations. Cytokine delivery using virus vectors and cell vehicles have been proposed to reduce systemic toxicity.
Genetically modified tumor cells releasing various cytokines have been shown to enhance tumor immunogenicity and to induce the regression of pre-existing tumors. Immunization with tumor cells modified to secrete IL-2 (Karp et al., 1993, J. Immunol. 150, 896-908), alpha interferon (IFNa) (Porgador et al., 1993, J. Immunol. 150, 1458-1470) or GM-CSF (Dranoff et al., 1993, PNAS 90, 3539-3543) have been shown to enhance tumor immunogenicity and to induce the regression of preexisting tumors. In some instances, immunological memory has been generated to resist the subsequent challenge with unmodified, parental tumor cells. Moreover, cytokine-transduced tumors may attract an inflammatory exudate in vivo that generally results in tumor destruction in animal models. Experimental animals and a small number of patients with established neoplasms treated with the cytokine-secreting tumor cells survived for a longer period of time, although in most instances tumor-growth eventually recurred.
The direct injection into solid tumors of vectors carrying genes encoding a variety of cytokines and chemokines has also been attempted in order to enhance the presentation of T-cell epitopes or to enhance the activation of tumor-specific T-lymphocytes. Many cytokines, including gamma interferon (IFN-g), IL-2 (Slos et al., 2001, Cancer Gene Ther. 8, 321-332), IL-7 (Miller et al., 2000, Human Gene Therapy 11(1), 53-65; Sharma et al., 1996, Cancer Gene Therapy 3, 302-313), IL-12 (Melero et al., 2001, Trends Immunol. 22, 113-115), IL-15 (Suzuki et al., 2001, J. Leukoc. Biol. 69, 531-537; Kimura et al., 1999, Eur. J. Immunol. 29, 1532-1542), IL-18 (Cao et al., 1999, FASEB J. 13, 2195-2202), and IL-21 (Ugai et al., 2003, Cancer Gene Therapy 10, 187-192) have demonstrated significant antitumor activity in mice. For example, intra-tumoral injection of dendritic cells transduced with an adenovirus expressing IL-7 leads to significant systemic immune responses and potent anti-tumor effects in murine lung cancer models (Miller et al., 2000, Hum Gene Ther. 11, 53-65).
More recently, many studies with both mouse and human tumor models have shown the importance of cytokine combinations in the development of optimal immune responses (see for example Putzer et al., 1997, Proc Natl Acad Sci USA. 94, 10889-10894; Melero et al., 2001, Trends Immunol. 22, 113-115; Zhu et al., 2001, Cancer Res. 61, 3725-3734). For example, the combination of IL-12 with the Th1 promoting IL-18 has been shown useful for the stimulation of the cell-mediated immune response (Hashimoto et al., 1999, J. Immunol. 163, 583-589; Barbulescu et al., 1998, J. Immunol. 160, 3642-3647). IL-2 and IFNg have been shown to cooperate for inhibiting tumor cell growth (U.S. Pat. No. 5,082,658). More recently, IL-21 was described to synergize the effects of IL-15 or IL-18 in the enhancement of IFNg production in human NK and T cells (Strengell et al., 2003, J. Immunol., 170, 5464-5469). The combination of IL-4 and GM-CSF is particularly useful in stimulating DCs (Palucka et al., 1998, J. Immunol. 160, 4587-4595). In other studies, it was found that the combination of IL-3 and IL-11 had a synergistic effect with IL-12 on the proliferation of early hematopoïetic progenitor cells (Trinchieri et al., 1994, Blood 84, 4008-4027). Graham and colleagues pioneered the combination of two adenoviruses, one encoding IL-2 and the other IL-12 (Addison et al., 1998, Gene Ther. 5, 1400-1409). They observed complete regression in more than 60% of established mammary carcinomas and induction of potent antitumor CTL activity. Recent data show that IL-15 can also synergize with IL-12 after double-transfection of human lung cancer cells (Di Carlo et al., 2000, J. Immunol. 165, 3111-3118). Also, IL-18 has been identified as a potent inducer of IFNg, and importantly, upregulates the expression of IL-12 receptors (Nakanishi et al., 2001, Ann. Rev. Immunol. 19, 423-474). In a reported poorly immunogenic tumor (MCA205), a clear synergy between these two cytokines was observed with antitumor effects mainly mediated by NK cells.
However, in many of these studies, it was found that the relative level of each cytokine was very important. For example, synergy studies between IL-12 and other cytokines for the generation of antitumor responses in mice have shown mixed results. Whereas the addition of IL-12 in the presence of suboptimal amounts of IL-2 led to synergy in the induction, proliferation, cytolytic activity and IFNg induction, combinations of IL-2 and IL-12 using a high dose of one cytokine were found to be antagonistic (Perussia et al., 1992, J. Immunol. 149, 3495-3502; Mehrotra et al., 1993, J. Immunol. 151, 2444-2452). In some models, a non-optimal dose of one cytokine with respect to the other led to an enhanced toxicity, while in other models, combinations of IL-12 and IL-2 showed little or no synergy (e.g., Nastala et al., 1994, J. Immunol. 153, 1697-1706). A similar situation occurs with combinations of IL-12 and IL-7. These results may reflect the inherent difficulty of combining two potentially synergistic cytokines in vivo, especially when there is a need to maintain a fixed ratio of activities of two components with different pharmacological properties, such as different circulating half life and biodistribution.
To reduce the difficulties inherent to cytokine combinations, one strategy is to fuse the cytokines. Fusions between two cytokines have already been proposed in the literature. For example, WO 01/10912 describes fusions between IL-12 and a second cytokine with short half life in order to provide a longer pharmacokinetic behavior similar to that of IL-12 itself. The fusion of IL-12 with either IL-2, granulocyte-macrophage colony-stimulating factor (GM-CSF) or IL-4 is specifically disclosed. U.S. Pat. No. 5,883,320 and WO 92/04455 disclose fusions between IL-3 and a second cytokine, which may be used in the treatment of diseases associated with a decreased level of hematopoietic cells. The fusion between IL-3 and IL-11 was shown to be useful for stimulating the production of megakaryocytes and platelets. Drexler et al. (1998, Leuk Lymphoma 29, 119-128) describe the fusion of GM-CSF and IL-3. Finally, U.S. Pat. No. 6,261,550 envisages the fusion of G-CSF with a cytokine to enhance hematopoïesis., e.g., to compensate hematopoietic deficits resulting from chemotherapy or radiation therapy in cancer patients.
The development of efficient molecules against human tumors has been a long sought goal which has yet to be achieved. In light of the forgoing, there remains a need for cytokine fusions which evoke an immune response and are capable of bypassing tumor immunosuppression.
This technical problem is solved by the provision of the embodiments as defined in the claims.